Technical Field
The present invention relates to a method for purifying an erythropoietin analog having a low isoelectric point below 4 by adding an N-linked sugar chain with high purity.
Background Art
Erythropoietin is an erythropoiesis-stimulating factor that promotes red blood cell production by stimulating hematopoietic stem cells and facilitating their differentiation into erythrocytes. Erythropoietin (or EPO) is a glycoprotein having three N-linked sugar chains and one O-linked sugar chain and is used to treat anemia in patients with chronic kidney disease, cancer and the like. The number of sialic acid residues in the sugar chains of EPO affects the half-life and biological activity of EPO in blood. An isoform having a larger number of sialic acid residues has higher biological activity as degradation of EPO is suppressed (Egrie J C. and Browne J K., Br. J. Cancer, 2001; 84, 3-10 and Egrie et al., Glycoconj. J., 1993; 10: 263-269).
Recently, a novel erythropoiesis-stimulating protein (NESP), which is an erythropoietin analog with a low isoelectric point obtained by adding N-linked sugar chains to naturally occurring erythropoietin through genetic modification in order to increase biological activity, has been produced. Darbepoetin alfa is an NESP currently available as drug (Egrie and Browne, Br. J. Cancer. 84 Suppl. 1, 3-10 (2001)). NESP is a glycoprotein consisting of 165 amino acids. Whereas naturally occurring EPO has up to 14 sialic acid residues, NESP has up to 18 or 22 sialic acid residues by added N-linked sugar chains. Sialic acid is a sugar acid. As the number of sialic acid residues increases as a result of addition of the N-linked sugar chains, whereby the NESP molecule has a lower isoelectric point (pI) (Rush R S, et al., Anal. Chem., 1995; 67: 1441-52).
Since the NESP obtained by addition of the sialic acid residues has 3 times longer half-life in serum as compared to the existing recombinant erythropoietin, equivalent therapeutic effect can be expected with fewer administrations when treating anemia of patients with chronic kidney disease. The sialic acid content of EPO is directly related with the drug release duration in vivo and thus is regarded as an important property of the glycoprotein. The sialic acid residue at the terminal of the sugar chain of EPO protects the second galactose residue of the sugar chain, thereby inhibiting the degradation of the galactose residue by the hepatocyte receptor, extending half-life of EPO in vivo and improving its biological activity (Goldwasser et al, J. Biol. Chem. 249, 4202-4206 (1974), Lowy et al., Nature. 185, 102-103 (1960)).
A method of producing EPO using animal cells is disclosed in the following patent. Korean Patent Registration No. 10-0423615 describes a method for producing erythropoietin by culturing EPO-producing cells on a serum-free flask or roller and purifying them sequentially by Blue Sepharose adsorption chromatography, hydrophobic chromatography, anion exchange chromatography and high-speed liquid chromatography. However, this patent does not describe a method for producing NESP, and the purification procedure involving the four chromatographic processes disclosed in the patent is complicated.
In addition, use of immunoadsorption chromatography is described in Korean Patent Registration Nos. 10-0153808 and 10-0900013, use of dye affinity chromatography is described in Korean Patent Registration Nos. 10-0390325 and 10-0423615, and use of reversed phase chromatography is described in Korean Patent Registration Nos. 10-0065197 and 10-0423615, Korean Patent Publication No. 10-2004-0065567 and Korean Patent Registration No. 10-0900033. Further, use of hydrophobic chromatography is described in Korean Patent Registration Nos. 10-0344059 and 10-0567448, and use of batch ion-exchange chromatography is described in Korean Patent Registration No. 10-0297927. Lastly, use of hydroxyapatite chromatography is described in Korean Patent Registration Nos. 10-0390325 and 10-0900013 and U.S. Pat. No. 7,619,073. Although the above-described techniques describe the purification of EPO, they do not describe purification of NESP having a lower isoelectric point than EPO.
Meanwhile, U.S. Pat. No. 7,217,689 disclosing NESP material describes use of anion exchange chromatography in the Examples.
WO2010/008823 A2 describes use of anion exchange or adsorption chromatography together with cation exchange chromatography for purification of NESP. In particular, this publication proposes a method for separating isoforms having more sialic acid residues through cation exchange chromatography.
As described, the purification method in the existing patents relating to EPO involves the use of columns inapplicable in industrial-scale production or the complicated purification processes of four or more steps. In addition, the method is not applicable for NESP having a lower isoelectric point owing to change in physicochemical properties due to the addition of sugar chains. The recently-disclosed sole patent relating to purification of NESP involves four steps, and its main process of separating isoforms having more sialic acid residues uses cation exchange chromatography. In contrast, anion exchange chromatography which is capable of more precisely separating the isoforms having more sialic acid residues is used in the present invention. Further, the present purification procedure is consisting of simpler three-step chromatographic processes, and it allows purification of erythropoietin analogs having a low isoelectric point faster at lower cost.
Throughout the specification, a number of publications and patent documents are referred to and cited. The disclosure of the cited publications and patent documents is incorporated herein by reference in its entirety to more clearly describe the state of the related art and the present disclosure.